This invention relates to a method for making neural network microelectrode arrays derived from passaged progenitor cells. The array may be used for various biosensing applications including detection and/or quantification of various biological, chemical or environmental threats, including toxins, such as neurotoxins, nerve agents, such as neurotransmitter antagonists and neurotransmitter inhibitors, bacterial agents, viral agents, choking agents, fuel and/or combustion products, toxic metals, herbicides and/or pesticides, bioregulators, psychogenic and/or hallucinatory drugs, n-acyl ethanolamines and other miscellaneous compounds.
Traditional biosensor technologies operate by detecting specific, known environmental threats. Recent technological advances have led to the development of more broad-spectrum generic detection methodologies to complement target specific sensors and assays. These generic biosensors utilize the functional responses of a biological system as sensor elements to detect and/or quantify both known and unknown toxins.
For example, neural network microelectrode arrays derived from primary neural tissue have been shown to detect a range of environmental toxins in various media such as samples of potable water and sea water. (Shaffer, K. M., Gray, S. A., Fertig, S. G., Selinger, J. V., O'Shaughnessy, T. J., Kulagina, N. V., Stenger, D. A., and Pancrazio, J. J., “Neuronal Network Biosensor for Environmental Threat Detection,” 2004 NRL Review, chemical/biochemical research, pp. 118-120, (2004); Kulagina, N., et al. “Pharmacological effects of the marine toxins, brevetoxin and saxitoxin, on murine frontal cortex neuronal networks,” Toxicon 44, pp. 669-676, (2004); Kulagina, N., et al. “Detection of Marine Toxins, Brevetoxin-3 and Saxitoxin, in Seawater Using Neuronal Networks,” Environ. Sci. Technol. 40, pp. 578-583, (2006); O'Shaughnessy, T. J., Gray, S. A., Pancrazio, J. J., “Cultured neuronal networks as environmental biosensors,” J. Appl. Toxicol. 24, pp. 379-385, (2004)). These arrays utilize networks of mammalian neurons as sensing elements. The networks are grown over thin-film microelectrode arrays (MEAs), which enable extracellular monitoring of bioelectrical activity over at least 64 MEA contact points (Gross, G. W., Harsch, A., Rhoades, B K., Gopel, W., “Odor, Drug, and Toxin Analysis with Neuronal Networks in Vitro: Extracellular Array Recording of Network Responses.” Biosens. Bioelectron. 12, 373, (1997): Pancrazio, J. J., Gray, S. A., Shubin, Y. S., Kulagina, N., Cuttino, D. S., Shaffer, K. S., Eisemann, K., Curran, A., Zim, B., Gross, G. W., and O'Shaughnessy, T. J., “A Portable Microelectrode Array Recording System Incorporating Cultured Neuronal Networks for Neurotoxin Detection.” Biosens. Bioelectron. 18, p. 1339, (2003)). Changes in action potential dynamics across the neural network. i.e. the rate at which action potentials are fired across the neural network, can be used as a basis for detecting neuron-active substances (Pancrazio, J. J., et al., “Development and Application of Cell-Based Biosensors.” Biomedical Engineering Society 27, pp. 697-722. (1999): Stenger, D. A., et al., “Detection of physiologically active compounds using cell-based biosensors.” Trends in Biotech, Vol. 19, no. 8, pp. 304-309, (2001)).
The action potentials observed in the networks are largely driven by the synaptic integrity of the network, rather than being the result of independent spiking neurons. Thus, compounds that affect the synapses dramatically affect the overall rate at which action potentials are fired across the network. Extracellular monitoring of bioelectrical activity from cultured networks grown over MEAs is a noninvasive method that enables long-term monitoring. The neural network MEA derived from primary neural tissue can potentially be configured as a portable instrument that requires only minimal training to operate (Pancrazio, J. J., Gray, S. A., Shubin, Y. S., Kulagina, N., Cuttino, D. S., Shaffer, K. S., Eisemann, K., Curran, A., Zim, B., Gross, G. W., and O'Shaughnessy, T. J., “A Portable Microelectrode Array Recording System Incorporating Cultured Neuronal Networks for Neurotoxin Detection.” Biosens. Bioelectron. 18, p. 1339, (2003)).
The primary neural tissue cultures used to generate these neural networks, however, are difficult to obtain and require the use of a significant number of animals in order to provide sufficient source material for the tissue cultures. Recently, scientists have been experimenting with stem cells and progenitor cells as an alternative, self-renewing resource capable of forming neural networks. Whereas primary cultures of neural stem cells and progenitor cells, which are isolated directly from developing animal brains, provide a limited amount of neural tissue, a single progenitor cell culture, which may be induced to form functional neural networks, can potentially regenerate for an indefinite period of time through cell passaging. Passaging cells may involve splitting cells by protease treatment and detaching the cells from the culture surface before the cell density becomes confluent. Trypsin is commonly used for detachment and such a splitting process can be repeated multiple times, however, eventually the cells stop proliferating due to other stresses. (Shaffer, K. M., Lin, H. J., Maric, D., Pancrazio, J., Stenger, D. A., Barker, J. L., Ma, W., “The use of GABAA receptors expressed in neural precursor cells for cell-based assays.” Biosensors & Bioelectronics, 16, pp. 481-489, (2001)).
U.S. Pat. No. 6,197,575 discloses a matrix seeded with undifferentiated cells, such as embryonic cells, stem cells and other precursor cells. The matrix may be used to detect biological toxins. U.S. Patent Publication no. 2004/0106168 also discloses a neural network grown on an MEA from primary neural tissue cultures. This document also suggests that 12-16 day old embryonic cells may be plated on the MEA to generate the neural network but does not exemplify such a method. Mitogenic growth factors can synergize with N-CAM or neurotrophins to generate spontaneously active neural networks on MEAs from neural progenitor cells. Mistry, S. K., Keefer, E. W., Cunningham, B. A., Edelman, G. M., Crossin, K. L., “Cultured rat hippocampal neural progenitors generate spontaneously active neural networks,” PNAS. 99, pp. 1621-1626, (2002). This method for generating neural networks from neural progenitor cells, which consists of disassociating embryonic stem cells from the hippocampal region, growing the cells in a serum free medium and plating the disassociated cells, is inefficient and produces a limited amount of neuroglia and neurons. Mistry, S. K., Keefer, E. W., Cunningham, B. A., Edelman, G. M., Crossin, K. L., “Cultured rat hippocampal neural progenitors generate spontaneously active neural networks.” PNAS. 99, pp. 1621-1626, (2002).
Although neural stem cells and progenitor cells are capable of self-renewal and differentiating into functional neural networks and, as such, can be utilized to mass produce cells for biosensor or pharmacologic testing, the natural differentiation process is complex and far from fully understood. Several approaches have been used to induce neural stem cell differentiation into functional neural networks. The majority of these methods focus on providing various culture conditions and means for regulating differentiation. Growth factors, neurotrophins and cell adhesion molecules have been shown to regulate neural stem cell differentiation (Panchision, D. M., McKay, R. D., “The control of neural stem cells by morphogenic signals.” Current Opinion Genetic Dev. 12, pp. 478-87, (2002): Hosomi S, Yamashita T, Aoki M, Tohyama M., “The p75 receptor is required for BDNF-induced differentiation of neural precursor cells.” Biochem Biophys Res Commun. 301, pp. 1011-5, (2003): and Zheng, W., Nowakowski, R. S., Vaccarino, F. M., “Fibroblast growth factor 2 is required for maintaining the neural stem cell pool in the mouse brain subventricular zone.” Dev Neurosci. 26, pp. 181-96, (2004)). Basic fibroblast growth factor (bFGF) is a growth factor that has been demonstrated to promote cell proliferation and neurogenesis (Ma, W., Liu, Q. Y., Maric, D., Sathanoori, R., Chang, Y. and Barker, J. L. “Basic FGF-responsive telencephalic precursor cells express functional GABAA receptor/Cl− channels in vitro,” J. Neurobiol, 35, pp. 277-286 (1998)). Neuotrophins, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), have been reported to up-regulate neural differentiation. Cell adhesion molecules, such as neural cellular adhesion molecules (N-CAM), also affect cell proliferation and differentiation. For example, bFGF and either N-CAM or BDNF have been used to generate active neural networks from primary cultures of neural progenitors isolated from the rat hippocampus. Mistry, S. K., Keefer, E. W., Cunningham, B. A., Edelman, G. M., Crossin, K. L., “Cultured rat hippocampal neural progenitors generate spontaneously active neural networks.” PNAS. 99, pp. 1621-1626, (2002).
Although regulation of neural progenitor cell differentiation oil MEAs has been studied, it has not been shown that a reliable, efficient and/or cost-effective source of neural progenitor cells can induce the formation of functional neural network on MEAs. Therefore there currently exists a need to develop an efficient and effective method for generating neural network microelectrode arrays in a manner that enables continuous cellular proliferation.